This application represents a continuation and expansion of the aims of the past project period for designing synthetic sialoside probes to investigate ligand binding functions of the siglec family of cell adhesion receptors. Members of the siglec sub-group of the Ig superfamily have in common an N-terminal V set Ig domain which confers the ability to bind to sialyloligosaccharide sequences (sialosides) of glycoprotein and glycolipid carbohydrate groups (glycans). The human and murine siglec families now comprise eleven and seven members respectively. In addition to the N-terminal Ig sialic acid binding domain, they each have 1-16 additional extra-cellular Ig domains, and a C-terminal membrane anchor, and a cytoplasmic domain. With the exception of two siglecs (sialoadhesin and myelin associated glycoprotein (MAG)), the cytoplasmic domains contain one or more immuno-receptor tyrosine inhibitory motifs (ITIM) and/or activation motifs (ITAM) typically found in co-receptors that regulate cell signaling. In the next project period we will continue our efforts to develop sialoside-based tools that will have utility in elucidating the role of siglec-ligand interactions. A major goal is to create high affinity multivalent sialoside probes that have a unique specificity for a single siglec, and are capable of binding to cell surface siglecs without the need to destroy the cis ligands that are known to 'mask' the binding of lower affinity probes. We will also employ the approach of sialic acid metabolic engineering to incorporate high affinity sialic acid analogs that can alter the affinity of siglec-ligand interactions and thereby exaggerate their role in siglec function. A major new direction is to identify cis and trans ligands of siglecs using metabolic engineering to install sialic acids containing aryl- azide substituents as a 'bait', allowing photo-affinity crosslinking of glycans bound to siglecs. Once cross- linked, the attached ligands will be 'fished-out' using anti-siglec antibodies and identified using standard methodologies. Since siglecs are widely expressed on white blood cells that comprise the innate and acquired immune system, understanding their roles may provide insights into regulating the immune system in human health and disease.